The present invention relates to new N-phenyl-amide and N-pyridylamide derivatives, to the methods of preparing these compounds, to the pharmaceutical compositions containing them and to their use as medicaments especially in the treatment of hyperlipidaemia and atherosclerosis.
It is known that lipid deposits, especially cholesterol deposits in blood vessels are responsible for the formation of atheroma plaques which are the cause of a variety of cardiovascular diseases; more precisely, atheroma is a form of atherosclerosis characterized by an excessive accumulation of lipids, in particular of cholesterol esters, in the wall of the vessels; it has recently been found that an enzyme, acyl Coenzyme A: Cholesteryl Acyl transferase (ACAT) is responsible for the esterification of cholesterol, and a correlation was found between the increase in the activity of this enzyme and the accumulation of cholesterol esters in the vascular wall; it is also known that dietary cholesterol is absorbed in free form and is then esterified by intestinal ACAT for release into the bloodstream in the form of VLDL (very low density lipids) and/or of chylomicrons.
While several ACAT inhibitors have been identified (see for example: EP 295 637, EP 415 413 or EP 497 201) the development of new ACAT inhibitors having improved therapeutic properties should be continued.
Attempts have been made to develop ACAT-inhibiting products capable of preventing intestinal absorption of dietary and bile cholesterol and of acting against the deposition of cholesterol esters in the wall of the vessels.
This search for ACAT inhibitors has led the inventors to develop a new family of N-phenylamide and N-pyridylamide derivatives and to find that these products manifest a potent vascular ACAT-inhibiting activity associated with an intense antihyperlipidaemic effect on various animal species.
These properties of the compounds of the invention make them particularly useful especially for the treatment of hyperlipidaemia and of athero-sclerosis.
The compounds of the invention have, more precisely, the formula: 
in which X is O, S or CH2;
R1 and R2, which may be identical or different, are hydrogen, (C1-C6)alkyl or (C3-C8)cycloalkyl, or alternatively R1 and R2, together with the carbon atom bearing them, form (C3-C8)cycloalkyl;
R3 is a (C6-C12)aryl optionally substituted with one or more Y radicals, which may be identical or different; or a 5- to 7-membered heteroaryl comprising 1 to 3 endocyclic heteroatoms chosen from O, S and N which is optionally substituted with one or more Y radicals, which may be identical or different;
Y is halogen, a (C1-C6)alkyl optionally substituted with one or more halogens, a (C1-C6)alkoxy optionally substituted with one or more halogens, a (C1-C6)alkylthio optionally substituted with one or more halogens, (C1-C7)acylamino, (C1-C3)acyloxy, hydroxyl, nitro, cyano, amino, (C1-C6)alkylamino, di-(C1-C6)-alkylamino, pyrrolidono, piperidino, morpholino, (C1-C4) alkylsulfonylamino, (C2-C5) alkoxycarbonyl, carboxyl, (C2-C6)alkylcarbonyl, carbamoyl, (C2-C5)alkylcarbamoyl, di-(C2-C5)aalkylcarbamoyl or (C1-C6)alkylsulfonyl;
R4 and R5, which may be identical or different, are a Y radical or alternatively a hydrogen atom;
Ar is one of the following groups A, B or C: 
T1 and T2, which may be identical or different, are halogen, (C1-C6)alkoxy, (C1-C6)alkylthio or (C1-C6) alkyl;
T is a hydrogen atom or (C1-C6) alkyl;
T3 and T4, which may be identical or different, are (C1-C6) alkyl, (C1-C6) alkoxy, (C1-C6) alkylthio, (C6-C12)arylthio, (C1-C6)alkoxycarbonyl, (C1-C6)alkyl-carbonyl, (C6-C12)arylcarbonyl or xe2x80x94(CH2)pxe2x80x94OR in which p is 1, 2, 3 or 4 and R is (C2-C3)alkyl,
R6 and R7 are each a hydrogen atom or alternatively
R6 and R7 together are a bond;
z is either
(i) the divalent group xe2x80x94CHR9xe2x80x94 in which R9 is a hydrogen atom or (C1-C6)alkyl;
or (ii) the divalent group xe2x80x94CHR10xe2x80x94CHR11xe2x80x94 in which R10 and R11 together form a bond such that Z is xe2x80x94CHxe2x95x90CHxe2x80x94or alternatively R10 and R11, which may be identical or different, are as defined above for R9;
or (iii) the divalent group xe2x80x94CHR12xe2x80x94CHR13xe2x80x94CH2xe2x80x94 in which R12 and R13 together form a bond such that Z is xe2x80x94CHxe2x95x90CHxe2x80x94CH12xe2x80x94, or alternatively R12 and R13, which may be identical or different, are as defined above for R9, as well as their addition salts with a pharmaceutically acceptable acid or base.
The addition salts of these compounds with pharmaceutically acceptable acids or bases also form part of the invention. Examples of these salts are the salts formed from hydrochloric acid, p-toluenesulfonic acid, fumaric acid, citric acid, succinic acid, salicylic acid, oxalic acid, hydrobromic acid, phosphoric acid, methanesulfonic acid, tartaric acid and mandelic acid.
In some cases, the compounds of the invention have one or more chiral centres. It should be understood that each stereoisomer forms part of the invention.
(C1-C6)Alkyl is a linear or branched, saturated hydrocarbon radical of 1 to 6 carbon atoms. The (C1-C6)alkoxy group consequently is the alkyl-Oxe2x80x94 group and the (C1-C6)alkylthio group is the alkyl-Sxe2x80x94 group where alkyl is as defined above.
Moreover, (C3-C8)cycloalkyl is understood to mean a saturated mono- or bicyclic hydrocarbon radical comprising from 3 to 8 carbon atoms. Examples are cyclopropyl, cyclohexyl, cyclopentyl and cycloheptyl.
The term (C6-C12)aryl is, moreover, a mono- or polycyclic aromatic group having 6 to 12 carbon atoms, such as phenyl, naphthyl or anthryl. Thus, (C6-C12)arylthio is the (C6-C12)-aryl-Sxe2x80x94 radical.
As a 5- to 7-membered heterocycle comprising 1 to 3 endocyclic heteroatoms chosen from O, S and N, there may be mentioned furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, pyridine, pyridazine, pyrimidine and pyrazine.
The halogen atoms are chlorine, bromine, fluorine and iodine.
The term acyl is the alkylcarbonyl radical. Thus, (C1-C7)acylamino is (C1-C7)alkylcarbonylamino and (C1-C3)acyloxy is (C1-C3)alkylcarbonyloxy.
Among these compounds, there are 6 subgroups of preferred compounds.
A first subgroup consists of compounds of formula I in which Y is halogen, (C1-C6)alkyl, (C1-C6)alkoxy or trifluoromethyl.
A second subgroup comprises the compounds of formula I in which:
R1 and R2, which may be identical or different, are hydrogen or alternatively R1 and R2, together with the carbon atom bearing them, form (C3-C8)cycloalkyl;
R3 is a (C6-C12)aryl optionally substituted with one or more Y radicals, which may be identical or different;
Y is halogen;
R4 and R5 are each a hydrogen atom;
Ar is one of the following groups A, B or C: 
T1 and T2, which may be identical or different, are (C1-C6) alkyl;
T is a hydrogen atom or (C1-C6)alkyl;
T3 and T4, which may be identical or different, are (C1-C6)alkyl; (C1-C6)alkoxy or (C1-C6)alkylthio;
R6 and R7 are each a hydrogen atom or alternatively R6 and R7 together are a bond;
Z is either
(i) the divalent group xe2x80x94CHR9xe2x80x94 in which R9 is a hydrogen atom or (C1-C6)alkyl; or
(ii) the divalent group xe2x80x94CHR10xe2x80x94CHR11xe2x80x94 in which R10 and R11 together form a bond such that Z is xe2x80x94CHxe2x95x90CHxe2x80x94, or alternatively R10 and R11 are each a hydrogen atom.
A third subgroup consists of the compounds of formula I in which Z is xe2x80x94CHR12xe2x80x94CHR13xe2x80x94CH2xe2x80x94, R12 and R13 being as defined above.
Among the compounds of the first, second and third subgroups defined above, those for which R1 and R2 are a hydrogen atom are more particularly preferred.
A fourth subgroup consists of the compounds of formula I in which X is O or S and R1 and R2, together with the carbon atom bearing them, form a (C3-C8) cycloalkyl.
A fifth subgroup of preferred compounds comprises the compounds of formula I in which X is O or S and Z is xe2x80x94CHxe2x95x90CHxe2x80x94 or alternatively xe2x80x94CHxe2x95x90CHxe2x80x94CH2.
In general, it is preferable that Ar is 2,4-dimethylthio-6-methyl-3-pyridyl; 2-methoxy-4-hexylthio-3-pyridyl and 2,6-diisopropylphenyl.
A sixth subgroup consists of the compounds of formula I in which X is CH2.
Among these compounds, those for which Ar is a group B or C are more particularly preferred. Here again, the meanings 2,4-dimethylthio-6-methyl-3-pyridyl and 2-methoxy-4-hexylthio-3-pyridyl for Ar are particularly advantageous.
According to a preferred embodiment of the invention, R3 is preferably phenyl which is optionally substituted, pyridyl or thienyl which is optionally substituted, such as for example 2-pyridyl or 2-thienyl which is optionally substituted at the 5-position.
The compounds of the invention may be prepared by coupling an acid of formula II 
in which R1, R2, R3, R4, R5, R6, R7 and Z are as defined in claim 1, with an aromatic amine of formula III:
Arxe2x80x94NH2xe2x80x83xe2x80x83(III)
in which Ar is as defined above.
This method, as well as the preferred variants of this method which are described below, are a subject of the invention.
The coupling of the acid of formula II with the amine of formula III may be simply carried out by reacting the amine of formula III with an activated derivative of the acid of formula II such as an acid chloride, an ester or a mixed anhydride.
More precisely, persons skilled in the art know that they can envisage the amination of the following activated acid derivatives: Poxe2x80x94COxe2x80x94SH, Poxe2x80x94COxe2x80x94SR, Poxe2x80x94COxe2x80x94Sexe2x80x94Me, Poxe2x80x94COxe2x80x94B(OR)2, (Poxe2x80x94COO)4Si, Poxe2x80x94COxe2x80x94C (hal)3 or Poxe2x80x94COxe2x80x94-N3 in which
Po is: 
hal is a halogen atom, and
R is (C1-C6)alkyl.
The methods of activating organic acids are known in the art.
Moreover, the coupling of the acid of formula II with the amine III may be carried out using any of the techniques used in liquid-phase peptide synthesis.
These techniques are, for example, described in xe2x80x9cMethods of Peptide Synthesisxe2x80x9d T. Wieland and H.
Determann, Angew. Chem. Interm. Ed. Engl., 2, 358, (1963).
By way of example, the chlorides of the acid of formula II may be obtained by the action of SOCl2, oxalyl chloride, PCl3 or PCl5.
It is also possible to prepare an acid chloride by the action of triphenylphosphine, in carbon tetrachloride, on the acid of formula II.
For the preparation of an acid bromide, the corresponding brominated reagents, such as oxalyl bromide, PBr3 or PBr5, may be used.
As an example of preparation of a mixed anhydride, there may be mentioned the action of bis(2-oxo-3-oxazolidinyl)phosphinic acid on the acid of formula II. This reaction is preferably carried out in the presence of a base as in the majority of activating reactions. This base may be either pyridine, ethylenediamine or 4-dimethylaminopyridine.
Thus, according to a preferred embodiment of the invention, the compounds of formula I are prepared:
using the following steps (i) and (ii):
(i) an acid of formula II is treated with oxalyl chloride in the presence of dimethylformamide; and then
(ii) an amine of formula III is reacted with the compound obtained in step (i);
or alternatively
using the following steps (i) and (ii):
(i) an acid of formula II is treated with bis(2-oxo-3-oxazolidinyl)phosphinic acid in the presence of a base; and then
(ii) an amine of formula III is reacted with the compound obtained in step (i).
The following two operating protocols can for example be used for coupling the acid II with the amine III.
Method A:
According to this method, the acid of formula II is activated in the form of an acid chloride before being coupled to the amine III.
The reaction of oxalyl chloride with the acid of formula II is carried out in an apolar aprotic solvent such as a hydrocarbon, for example a halogenated hydrocarbon.
The oxalyl chloride and a catalytic quantity of dimethylformamide are added to a solution of the compound of formula II, kept at a temperature of between 15 and 25xc2x0 C. and preferably at room temperature. The reaction medium is then heated to a temperature of between 30 and 70xc2x0 C., for example the reflux temperature of the solvent used. The reaction is monitored by thin-layer chromatography. The solvent is then evaporated and the residue is taken up in an apolar aprotic solvent such as, for example, the halogenated hydrocarbon previously used, before being supplemented with the aromatic amine III and with a base such as pyridine or 4-dimethylaminopyridine. This reaction is continued for the length of time necessary at a temperature of between 15 and 85xc2x0 C., preferably at room temperature.
Method B:
According to this method, the acid of formula II is activated in the form of a mixed anhydride before being coupled to the amine III.
A weak base such as triethylamine is added to a solution of the acid of formula II in an apolar aprotic solvent such as a halogenated hydrocarbon, and then the reaction medium is heated to a temperature of between xe2x88x9210 and 10xc2x0 C., preferably between 0 and 5xc2x0 C. Bis(2-oxo-3-oxazolidinyl)phosphinic acid chloride is then added. When the reaction is complete, the aromatic amine of formula III is added to the reaction medium all at once, the latter being kept between xe2x88x9210 and 10xc2x0 C. (preferably between 0 and 5xc2x0 C.). A base in solution in an apolar aprotic solvent such as a halogenated hydrocarbon is then introduced in small portions to the reaction medium.
The compound of formula I obtained is then isolated and purified.
The amines of formula III are either directly available commercially, or are easily available from commercial products.
In the remainder of the text, methods of preparing the compounds of formula II are provided.
The compounds of formula II in which Z is xe2x80x94CHR9xe2x80x94 may be obtained by following the reaction scheme A. 
The first step allows the introduction of the carboxaldehyde functional group.
A compound of formula VIII in which R1, R2, R3, R4 and R5 and X are as defined above is reacted with phosphorus oxychloride. This reaction takes place in a preferably polar aprotic solvent such as dimethylformamide (DMF). The reaction temperature varies between xe2x88x9220xc2x0 C. and room temperature. Preferably, the reaction is carried out between 0xc2x0 C. and 5xc2x0 C. and its progress is monitored by thin-layer chromatography. The aldehyde VII obtained is isolated in the usual manner by dilution of the reaction medium in a water-ice mixture, neutralization and then extraction and purification.
The next step of reduction of the aldehyde functional group to a hydroxymethyl functional group is carried out using any of the methods known in the art, as long the reaction conditions are such that they do not cause undesirable side reactions. Where appropriate, the reactive functional groups of the groups R1, R2, R3, R4 and R5 are protected.
Among the reagents commonly used to this end, there may be mentioned lithium aluminum hydride, sodium borohydride or sodium cyanoborohydride. When sodium borohydride is used, the reaction is preferably carried out in a methanol-water mixture at a temperature of between xe2x88x9240 and 0xc2x0 C., better still between xe2x88x9225 and xe2x88x9215xc2x0 C. Here again, the compound obtained is isolated in a manner known per se.
The alcohol of formula VI thus isolated is then converted to the corresponding alkyl chloride. This conversion may be carried out in any manner, as long as the reaction conditions are such that they do not cause side reactions. Where appropriate, the reactive functional groups of the groups R1, R2, R3, R4 and R5 are protected.
A known method consists in treating the alcohol VI with thionyl chloride in an inert solvent such as, for example, a toluene- or benzene-type aromatic hydrocarbon, at a temperature of between 15 and 30xc2x0 C., preferably at room temperature.
Other reagents may be used for the chlorination of the compound VI such as, for example, PCl5, PCl3 or POCl3.
The chlorinated compound of formula V is then treated with an alkali metal cyanide (MCN) such as sodium cyanide in a polar aprotic solvent such as DMF. The reaction temperature is kept between 0 and 50xc2x0 C. depending on the reactivity of the chloride V. When MCN is sodium cyanide, a temperature of between 20 and 25xc2x0 C. is generally suitable. The compound of formula IV obtained is isolated and purified in a conventional manner.
The compounds of formula II in which R9 is hydrogen are easily prepared from the nitrile IV by acidic or basic treatment. To this end, the following reagent systems may be used:
NaOH/H2O2 or NaOHaq
H2SO4 
HCOOH/HBr or HCl
AcOH/BF3 
AcOH/HCl.
For example, the nitrile IV may be hydrolysed using an AcOH/HCl: 40/60 to 60/40 mixture, a 1/1 mixture being perfectly appropriate. In this case, the AcOH/HCl mixture preferably plays the role of solvent, the temperature being unimportant between 0 and 50xc2x0 C., preferably between 15 and 25xc2x0 C.
In order to obtain the compounds of formula I in which R9 is (C1-C6)alkyl, the corresponding compound of formula II in which R9 is a hydrogen atom is treated with an alkyl halide of formula R9-X in which X is a halogen atom, a group (C1-C6)alkylsulfonyloxy or (C6-C10)arylsulfonyloxy optionally substituted with (C1-C6)alkyl, and R9 is (C1-C6)alkyl, in the presence of a strong base capable of removing the hydrogen at the xcex1 position with respect to the carboxyl functional group in the compound of formula II (R9=H). Such a base is, for example, lithium diisopropylamide (LDA).
According to a preferred embodiment, LDA is prepared in situ from n-butyllithium and diisopropylamine at a temperature of between xe2x88x9215 and 5xc2x0 C., preferably at about 0xc2x0 C. The solvent used for the generation of LDA is a polar aprotic solvent such as tetrahydrofuran. The halide R9-X and the compound of formula II are then added to the reaction medium. The reaction temperature is, for example, a temperature of between 15 and 35xc2x0 C., preferably a temperature of between 20 and 25xc2x0 C.
When the compound of formula I is such that Z is xe2x80x94CHR10xe2x80x94CHR11xe2x80x94, it may be prepared according to reaction scheme B. 
The introduction of a bromine atom into the compound of formula VIII is obtained by the action of N-bromosuccinimide (NBS) on the compound of formula VIII dissolved in a polar aprotic solvent such as dimethylformamide in the absence of moisture. The reaction temperature is for example room temperature. It may nevertheless vary, depending on the reactivity of the compound of formula VIII, between 10 and 35xc2x0 C.
The next step consists in converting the brominated derivative obtained of formula IX to a compound of formula X. To do this, an alkyl acrylate of formula H2Cxe2x95x90CHxe2x80x94COOR in which R=(C1-C6)alkyl is reacted with the brominated derivative IX in the presence of palladium acetate, a phosphine and a base. The reaction advantageously takes place in a polar aprotic solvent such as dimethylformamide.
The base may be triethylamine, pyridine or 4-dimethylaminopyridine, preferably triethylamine.
The phosphine for example has the formula PArxe2x80x23 in which Arxe2x80x2 is preferably a C6-C12aryl optionally substituted with a (C1-C6)alkyl. PArxe2x80x23 is for example triphenylphosphine or tritolylphosphine.
For the reaction to progress well, the compound of formula IX, dissolved in DMF, the base, the phosphine and the palladium acetate are first brought into contact, then the acrylate of formula CH2xe2x95x90CHxe2x80x94COOR is added to the reaction medium.
The resulting ester of formula X is isolated in a conventional manner and then saponified in a manner known per se to give a compound of formula II in which R10 and R11 together form a bond.
Starting with this compound, it is possible to easily have access to all the compounds of formula II in which Z is xe2x80x94CHR9xe2x80x94CHR10xe2x80x94.
For example, the acid of formula II obtained above: 
is subjected to a catalytic hydrogenation. By judiciously controlling the hydrogenation conditions, there is obtained either a compound of formula II in which R6, R7, R10 and R11 are each a hydrogen atom, or a compound of formula II in which R6 and R7 together form a bond and R10 and R11 are each a hydrogen atom.
The compounds of formula II in which Z is xe2x80x94CHR12xe2x80x94CHR13xe2x80x94CH2xe2x80x94 may be obtained using a Wittig reaction starting with the aldehyde of formula VII (Scheme A). It is for example possible to use a reagent system composed (i) of a phosphonium halide of formula ROOCxe2x80x94CH2xe2x80x94CH2xe2x80x94P+A3, hale in which R is hydrogen or (C1-C6)alkyl, hal is halogen and A is chosen from a (C6C12)aryl optionally substituted with (C1-C6)alkyl and (ii) of a base such as an alkali metal tert-butoxide (tBuOK), an alkali metal hydride (NaH) or an alkyl-lithium (C4H9Li). The reaction may advantageously be carried out in a polar aprotic solvent such as dimethylformamide or tetrahydrofuran at a temperature of between 0 and 30xc2x0 C.
According to another of its aspects, the invention relates to a pharmaceutical composition comprising at least one compound of formula I, in combination with one or more pharmaceutically acceptable vehicles.
The vehicles which may be used are, for example, fillers, diluents, binders, wetting agents, disintegrating agents, surface-active agents, and lubricants. The pharmaceutical composition may be in any desirable unit form, including tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, injections (solutions and suspensions) and the like.
To prepare tablets, it is possible to use vehicles known in this field, for example excipients, such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and the like; binding agents, such as water, ethanol, propanol, a simple syrup, a glucose solution, a starch solution, a gelatin solution, carboxymethylcellulose, gum lac, methyl cellulose, potassium phosphate or polyvinylpyrrolidone and the like; disintegrating agents, such as dried starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyoxyethylenesorbitan, sodium lauryl sulfate, a stearic acid monoglyceride, starch, lactose and the like; disintegration inhibitors, such as refined sugar, stearin, cocoa butter, hydrogenated oils and the like; absorption accelerators, such as a quaternary ammonium base, sodium lauryl sulfate and the like, wetting agents, such as glycerin, starch and the like; adsorbing agents, such as starch, lactose, kaolin, bentonite, colloidal silicic acid and the like; a lubricating agent such as purified talc, salts of stearic acid, powdered boric acid, polyethylene glycol and the like.
In the case of the preparation of tablets, the tablets may, moreover, be coated with a customary coating material so as to be converted to sugar-coated tablets, tablets coated with a gelatin film, tablets bearing enteric coatings, film-coated tablets, or tablets with a double layer or with multiple layers.
To form into pills, it is possible to use, for example, known vehicles which are commonly used in this field, such as excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, kaolin or talc and the like; binders, such as powdered gum arabic, powdered gum tragacanth, gelatin, ethanol and the like; and disintegrating agents, such as laminaria powder, agar and the like.
To form suppositories, it is possible to use known vehicles which are widely used in this field, for example polyethylene glycols, cocoa butter, higher alcohols, higher alcohol esters, gelatin, semi-synthetic glycerides and the like.
To produce injectable preparations, solutions and suspensions are sterilized and they are preferably made isotonic with respect to blood. To produce injectable preparations, it is also possible to use vehicles which are commonly used in this field, for example water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, fatty acid esters of polyoxyethylenesorbitan and the like. In this case, an appropriate quantity of sodium chloride, glucose or glycerin may be added to the desirable pharmaceutical preparations in order to make the solution isotonic. Furthermore, it is possible to add to the desirable pharmaceutical preparations, where appropriate, dissolving agents, buffer solutions, analgesic agents which are customarily used, as well as colouring agents, preservatives, perfumes, taste-modifying agents, sweetening agents and other medicaments.
The compounds of the invention have proved to be potent inhibitors of acyl-coenzyme A. As such, they are useful in the treatment or the prophylaxis of hypercholesterolaemia, atheromatous atherosclerosis and can even prevent possible ischaemic accidents such as, for example, a myocardial infarction as well as cerebrovascular diseases.
The pharmacological properties of the invention compounds were demonstrated by the following tests.
Test A: measurement of in vitro hepatic ACAT inhibition in rats: male Wistar rats weighing 220-250 g were sacrificed by cervical dislocation; the liver was removed and homogerused to prepare the microsomal fraction by ultracentrifugation; these microsomes were incubated with 14C-oleyl coenzyme A according to the method described by P. J. GILLIES et al., Exp. and Mol. Pathol. 1986, 44, 329-339; lipids were extracted from the incubate with a methanol-chloroforme mixture and 14C-oleyl cholesterol was separated by TLC; the latter represented the measurement of the ACAT activity and the results were expressed in inhibitory concentration 50 (IC50) representing the concentration of compound inhibiting the ACAT activity by 50%.
As an example, the IC50 values of compounds No. 1, 4 and 6 were respectively 94xc3x9710xe2x88x929 mole/l, 74xc3x9710xe2x88x929 mole/l and 31xc3x9710xe2x88x929 mole/l.
Test B: measurement of intestinal absorption of cholesterol in rats; male Wistar rats weighing 230-250 g and fasted for 24 hours received simultaneously the test substance per os and triton WR 1339 by IV route; one hour later, they were again treated orally with 3H-cholesterol; three hours later, I ml of blood was taken from the retro-orbital sinus: the blood radioactivity determined on 0.1 ml of serum represented the measurement of the absorption of 3H-cholesterol. The results were expressed in effective dose 50 (ED50) in mg per kg of animal and represented the quantity of compound inhibiting the intestinal absorption of cholesterol by 50%.
As an example, the ED50 values of compounds No. 1, 4 and 6 were respectively 0.005 mg/kg, 0.038 mg/kg and 0.023 mg/kg.
Test C: hypercholesterolemia model; claim 1 compounds were tested by oral route in animals subjected to a cholesterol-rich diet;
As an example, in the male Wistar rat fed a 2.5% cholesterol enriched diet for 8 days and treated for 2 days with compound No. 1, total cholesterol was lowered by 50% at the dose of 0.78 mg/kg; the effect was mainly observed on VLDL (Very Low Density Lipid).
As an example, in the rabbit fed a 0.5% cholesterol enriched diet for 15 days and treated sumultaneously with compound No. 1, total cholesterol was decreased by 70% at the dose of 0.1 mg/kg; the effect was mainly observed on VLDL (Very Low Density Lipid).